Stem cell factor attenuates vascular smooth muscle apoptosis and increases intimal hyperplasia after vascular injury.

OBJECTIVE: Stem cell factor (SCF) through its cognate receptor, the tyrosine kinase c-kit, promotes survival and biological functions of hematopoietic stem cells and progenitors. However, whether SCF/c-kit interactions exacerbate intimal hyperplasia through attenuating VSMC apoptosis induced by vascular injury has not been thoroughly investigated. METHODS AND RESULTS: VSMCs were stimulated with serum deprivation and H2O2 to induce apoptosis. The transcription of c-kit mRNA and the expression of the c-kit protein by VSMCs were estimated by Q-polymerase chain reaction and Western blotting, respectively. The interactions of SCF and c-kit were investigated by in vitro and in vivo experiments. In vitro, H2O2 stimulation significantly induced apoptosis of VSMCs as evidenced by the 3- and 3.2-fold increases of cleaved caspase-3 compared with those in the control group by Western blot and flow cytometric analyses, respectively (P<0.01). Stimulation of apoptosis also caused 3.5- and 9-fold increases in c-kit mRNA transcription and protein expression, respectively, by VSMCs compared with those in the control group. Administration of SCF (10 to 1000 ng/mL) significantly lowered the amount of cleaved caspase-3 in H2O2-treated VSMCs (P<0.01). Specifically, SCF exerted this effect through activating Akt, followed by increasing Bcl-2 and then inhibiting the release of cytochrome-c from the mitochondria to the cytosol. In vivo, the mouse femoral artery was injured with a wire in SCF mutant (Sl/Sl(d)), c-kit mutant (W/W(v)), and colony control mice. In colony control mice, confocal microscopy demonstrated that the wire-injury generated a remarkable activation of caspase-3 on medial VSMCs, coinciding with upregulation of c-kit expression. The wire-injury also caused an increase in the expression of SCF on surviving medial VSMCs and cells in the adventitia. The upregulated c-kit expression in the vessel wall also facilitated homing by circulating SCF+ cells. Compared with colony control mice, vascular injury in SCF mutant and c-kit mutant mice caused a higher number of apoptotic VSMCs on day 14 and a lower number of proliferating cells, and resulted in significantly less neointimal formation (P<0.01) on day 28. CONCLUSIONS: The interactions between SCF and the c-kit receptor play an important role in protecting VSMCs against apoptosis and in maintaining intimal hyperplasia after vascular injury.

Pioglitazone increases the numbers and improves the functional capacity of endothelial progenitor cells in patients with diabetes mellitus.

BACKGROUND: Endothelial progenitor cells (EPCs) are present in peripheral blood and can promote postnatal angiogenesis. The number and function of circulating EPCs are altered in diabetics. This study sought to investigate whether the number and functional properties of EPCs from patients with type II diabetes could be improved by pioglitazone. METHODS: For this randomized controlled study, we recruited 36 type II diabetic patients on metformin monotherapy with a glycohemoglobin A1c of <7%. Patients were separated into pioglitazone (n = 24) and control (n = 12) groups. The number and functional activity of EPCs, and the brachial artery flow-mediated dilation were determined before and after pioglitazone treatment (8 weeks) as an add-on therapy to metformin. In addition, direct effects of pioglitazone on EPCs were also investigated. RESULTS: After pioglitazone treatment, the numbers of circulating EPCs significantly increased (from 0.44% +/- 0.14% to 0.89% +/- 0.29%, P = .01). The migratory response and the adhesive capacity to fibronectin and collagen were improved by 158%, 34%, and 83%, respectively (all P < .05). Treatment with pioglitazone significantly lowered triglyceride, very low density lipoprotein cholesterol, and high-sensitivity C-reactive protein (hsCRP) levels, and increased high-density lipoprotein levels and insulin sensitivity (all P < .05). The increase in the number of circulating EPCs and the improvement in the migratory response after pioglitazone treatment were independently correlated to the decrease in hsCRP levels (P < or = .01). The increase in the adhesive capacity was independently correlated to the decreases in very low density lipoprotein cholesterol (P = .01) and hsCRP levels (P = .03). In addition, pioglitazone was also demonstrated to have direct effects on increasing EPC proliferation and colony formation, and attenuating EPC apoptosis (all P < .05, versus the controls). There were no significant changes in flow-mediated dilation in either group. CONCLUSIONS: Pioglitazone significantly increased the number and improved the functional properties of EPCs in type II diabetic patients through direct effects and/or anti-inflammation and lipid modification effects.

Tetrahydrobiopterin deficiency exaggerates intimal hyperplasia after vascular injury.

Decreased levels of tetrahydrobiopterin (BH4), an absolute cofactor for nitric oxide synthase (NOS), lead to uncoupling of NOS into a superoxide v. nitric oxide producing enzyme, and it is this uncoupling that links it to the development of vascular disease. However, the effects of in vivo deficiency of BH4 on neointimal formation after vascular injury have not been previously investigated. Hph-1 mice, which display 90% deficiency in guanine triphosphate cyclohydrolase I, the rate limiting enzyme in BH4 synthesis, were used. Hph-1 and wild-type mice, treated with either vehicle or BH4 (n = 15 per group), were subjected to wire-induced femoral artery injury, and NOS expression and activity, inflammation, cell proliferation, superoxide production, and neointimal formation were assessed. The major form of NOS expressed over vessel wall after vascular injury was endothelial NOS. Hph-1 mice exhibited lower NOS activity (2.8 +/- 0.3 vs. 4.5 +/- 0.4 pmol/min/mg protein, P < 0.01), and higher aortic superoxide content (5.2 +/- 2.0 x 10(5) cpm vs. 1.6 +/- 0.7 x 10(5) cpm, P < 0.01) compared with wild-type controls, indicating uncoupling of NOS. Treatment of hph-1 mice with BH4 significantly increased NOS activity (from 2.8 +/- 0.3 to 4.1 +/- 0.4 pmol.min(-1).mg protein(-1), P < 0.05), and attenuated superoxide production (from 5.2 +/- 2.0 x 10(5) cpm to 0.8 +/- 0.7 x 10(5) cpm, P < 0.05). Hph-1 mice also had higher inflammatory reactions and more cell proliferation after vascular injury. Furthermore, hph-1 mice responded by a marked increase in neointimal formation at 4 wk after vascular injury, compared with wild-type controls (intima:media ratio: 4.5 +/- 0.5 vs. wild-type 0.7 +/- 0.1, P < 0.001). Treatment of hph-1 mice with BH4 prevented vascular injury-induced increase in neointimal formation (intima:media ratio: 1.4 +/- 0.1 vs. hph-1, P < 0.001). Treatment had no effect on wild-type controls. In summary, we describe, for the first time, that in vivo BH4 deficiency facilitates neointimal formation after vascular injury. Modulation of BH4 bioavailability is an important therapeutic target for restenosis.